1. Field of the Invention
A method for enhancing the effectiveness of vaccines in animals, by maintaining the animals on a diet of contamination-resistant feed.
2. Discussion of the Background
Livestock such as poultry, swine, cattle and horses are routinely treated with vaccines to prevent viral and bacterial diseases. Breeders are sometimes vaccinated for another reason, to provide passive immunity to their offspring by supplying antibodies through colostrum in the case of mammals, and through egg yolk in the case of birds. Because neonatal animals have underdeveloped immune systems, passive immunity is their main source of protection against viral and bacterial diseases.
It is known that animal feed can be rendered highly resistant to contamination by pathogens, by spraying it with formaldehyde using an atomizing spray technique disclosed by Bland et al., U.S. Pat. No. 5,505,976 and divisional U.S. Pat. No. 5,591,467, both incorporated herein by reference. It was also disclosed that animals maintained on such feeds are more productive, in terms of feed conversion efficiency. It has now been discovered that animals which are maintained on a diet of contamination-resistant feedstuffs respond more strongly when immunized against diseases, with anti-viral vaccines and anti-bacterial vaccines (bacterins). Also, transference of passive immunity to offspring is enhanced by maintaining a breeder on contamination-resistant feedstuffs, providing a method for improving the health of neonatal animals.
An object of the invention is to provide a method for improving the immune response of an animal to a vaccine by maintaining it on a diet of contamination-resistant feed and treating the animal with an anti-viral or anti-bacterial vaccine.
Another object of the invention is to provide a method for increasing the level of antibodies in eggs, colostrum or milk produced in response to vaccination, by maintaining a breeding animal on a diet of contamination-resistant feed, and treating the breeding animal with an anti-viral or anti-bacterial vaccine.
Another object is to improve the absorption of antibodies by neonatal animals from egg yolk, colostrum or milk, by maintaining a breeding animal on a diet of contamination-resistant feed, vaccinating the breeder with an effective amount of an anti-viral or anti-bacterial vaccine and administering the resulting egg yolk, colostrum or milk to a neonatal animal.
When an animal is maintained on a diet of contamination-resistant feedstuffs, produced by treatment with formaldehyde in accordance with the methods of Bland et al., subsequent vaccinations of the animal with standard anti-viral and anti-bacterial vaccines are more effective because the amount of antibodies produced by the animal increases significantly. In the case of breeding animals the amount of antibodies deposited in the egg yolk, milk or colostrum also increases, which is of benefit to the offspring.
Applying formaldehyde to animal feed to kill Salmonella was known before the method of Bland et al., however, previously the goal was merely to provide an immediate killing effect. Formaldehyde solutions were mixed thoroughly with the feed in sufficient quantity to kill the Salmonella, using the same spray equipment ordinarily used to apply mold inhibitors. Such spraying equipment is designed to produce a coarse spray, i.e., droplet sizes in the range 260-400 microns, to decrease energy requirements and increase the application rate. The need to distribute formaldehyde throughout the feed was understood, because more than 99% of the Salmonella should be killed to prevent it from quickly recontaminating the feed. Coarse spraying with large amounts of formaldehyde is adequate for that purpose. Bland et al. discovered that when aqueous formaldehyde is sprayed onto feed in the form of a mist, using an atomizing sprayer, the resulting feedstuff is substantially more resistant to recontamination by pathogenic bacteria than feed treated with the same quantity of formaldehyde using conventional spray nozzles. In each case all of the bacteria are killed immediately, but the misting method produces a strong residual killing effect. It was also observed that much less formaldehyde is necessary to obtain equivalent resistance levels, in terms of the time a sample can resist challenge under aerobic conditions with E. coli or Salmonella.
It is possible to characterize a difference in physical properties between contamination-resistant feeds used in the present invention and feeds treated with formaldehyde using conventional spray nozzles. The quantity and distribution pattern of a formaldehyde adduct in the feed can be measured using an acidic hydrolysis assay. A highly uniform distribution of adduct, expressed in terms of the coefficient of variation (CV), is related to the feed""s contamination resistance.
The formaldehyde adduct""s distribution pattern through the feed is controlled by the size of spray droplets used to apply the formaldehyde, the thoroughness and speed of mixing the feed during the application process, the rate at which the formaldehyde solution is applied, and the residence time of feed in the mixer. A compromise must be reached with regard to some conflicting variables. For instance, it is desirable to move feed through the mixer as quickly as possible for economic reasons, but too short a residence time results in inadequate mixing even if the flow rate of formaldehyde solution is increased. The residence time in a two-ton (1814.4 kgs) horizontal mixer is typically three to five minutes. The formaldehyde solution should be delivered at a rate of 20-40 gal/hr. (75.5-151.4 liters/hr.). The size of spray particles is preferably small, 20-80 microns. However this range limits flow rate and may require several nozzles. Larger spray sizes, up to about 250 microns, can also be effective if other variables are adjusted to compensate, such as increased mixing rate or residence time, increased amounts of formaldehyde solution, or accepting a decreased resistance to contamination of the finished feed. Typically the best compromise will be a spray particle size in the 10-200 micron range. Suitable application rates for 1 kg of solution per metric ton of feed span the range of 15-90 seconds, preferably 45-60 sec.
The coefficient of variation should be 7% or less to achieve significant resistance to pathogenic bacteria, preferably 5% or less. The term xe2x80x9cresistant to contamination by pathogenic bacteriaxe2x80x9d means that a challenge with 1000 colony forming units (CFU) per gram of feed results in the death of substantially all the bacteria within 24 to 72 hours. In particular, the term xe2x80x9cresistance to contamination by Salmonella or E. colixe2x80x9d means that a challenge with 1000 CFUs of Salmonella or E. coli per gram of feed results in 1 CFU or less per 25 grams of feed after 24 hours incubation at 25xc2x0 C. A value of 5% CV or less allows reduced quantities of formaldehyde to be maximally effective. Also, such uniform formaldehyde distribution results in much less emission of formaldehyde vapor from the freshly treated feed and appears to increase the yield of adduct. Bacterial resistance is a function of the coefficient of variation. At 7% CV, the product will have relatively low resistance, which also varies according to the quantity of formaldehyde applied. At 4 lbs. (1.81 kg) dry wt. of formaldehyde per metric ton of finished feed a 7% CV results in about 30 days resistance as measured by challenge with E. coli (1000 CFU/gram of feed). Conversely, at 2.0% CV and only 2 lbs. (0.91 kg) dry wt. of formaldehyde/metric ton, a finished feed will resist contamination by E. coli (1000 CFUs/gram of feed) for about 60 days.
The increased bacterial resistance of feedstuffs according to the invention can be seen in the following experiment reported in Bland et al., U.S. Pat. No. 5,505,976. A sterilized poultry starter mash (500 grams/treatment quantity level) was treated with 37 wt. % formaldehyde solution in quantities of 0.66, 1.33 and 2.00 pounds (0.30, 0.60 and 0.90 kg) dry weight of formaldehyde per ton (907.2 kg). One set of samples was treated with a coarse spray (270 microns) at 1.8 gal/hr. (6.81 liters/hr.) and another was treated with fine mist (43 microns) at 25.5 gal/hr. (96.5 liters/hr.). Each sample of 500 grams was challenged with 20 ml of a liquid inoculum of Escherishia coli ( greater than 106 CFU/ml) one week after treatment. The feed treated with a coarse spray quickly became contaminated with E. coli whereas all quantity levels of the 29. feed treated with a mist were free of detectable levels of E. coli within 48 hours of the inoculation and for another 60 days, after which the test was discontinued. An experiment using 20 ml of inoculum containing Salmonella (103 CFU/ml) for recontamination gave similar results.
Most types of bacteria can be recovered from feed for assay purposes by placing a representative feed sample in an isotonic solution, such as buffered phosphate or saline, and plating this solution on a selective microbiological media. The inoculated media is incubated to visualize the bacteria. Some bacteria, such as Salmonella, may require selective pre-enrichment and/or enrichment steps to recover low levels of bacteria and damaged or stressed cells prior to selective media plating.
Suitable major ingredients of animal feed include cereal grains such as corn, grain sorghum, wheat, barley, oats, vegetable protein meals and animal by product meals. Complete animal feed products can also be treated with a mist of aqueous formaldehyde to produce an animal feed of the present invention. A typical complete feed would be a mash or pelletized feed containing corn, soybean meal, minerals, vitamins and other micro ingredients having a total of 7-22% protein, 3-6% fat, 2-5% fiber and an energy value of 1300-3500 kcal/gram.
Many different contamination-resistant animal feedstuffs can be used in the present invention. They are scientifically formulated for the species and age of animal being fed. For example, a laying hen diet would have more calcium in it than would a broiler finisher diet. Production of the feed is usually done in a mixer where all ingredients are added by weight then mixed. The immune response of the relevant animals can be enhanced with formaldehyde-treated turkey grower feed, chick starter feed, sow feed, dairy cow feed, feed for fish, shrimps, eels, etc.
The quantity of formaldehyde applied to the feedstuff is in the range 0.20-4.0 pounds (0.09-0.18 kg) dry wt. of formaldehyde per ton (907.2 kg) of feed, preferably 0.66-1.32 pounds (0.30-0.60 kg). A feed major ingredient should contain from 100 to 1000 grams of formaldehyde adduct per ton (907.2 kg). A complete feed product should contain about 100-660 grams of adduct per ton (907.2 kg). Formaldehyde is commercially available in 37 wt. % aqueous solution. One gram of this solution contains 370 mg or 12.3 mmol of formaldehyde.
The preferred mist or atomized spray consists of droplets in the size range 10-250 microns, preferably 20-200 microns and most effectively 40-80 microns. Spray nozzles capable of delivering these droplet sizes are commercially available. The preferred nozzles are those in which the liquid is supplied to the nozzle under pressure and compressed air is mixed with the liquid to produce a completely atomized spray. The droplet size is a function of air pressure and liquid pressure, so that a single nozzle can provide different sprays if desired. Particle droplet size can be reduced by increasing the air pressure to liquid pressure ratio. Droplet sizes at the different ratios can be determined by laser optics techniques using e.g., model OAP 2D GA2 manufactured by Particle Measuring Systems, Boulder, Colo.
A sufficient number of nozzles should be used to cover the surface of the feed in the mixing vessel. Para-formaldehyde is an insoluble condensation product of formaldehyde that should be removed prior to application of the solution to avoid clogging the spray apparatus. The solution preferably contains a C1-C8 alcohol such as methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol or phenol in the range 5-15 wt. % to stabilize the formaldehyde.
The aqueous formaldehyde solution can contain additional ingredients conventionally used to preserve animal feed, such as 5-15 wt. % C1-C8 carboxylic acids or salts thereof including formic acid, acetic acid, propionic acid, butanoic acid, benzoic acid, sorbic acid and lactic acid. Suitable salts include alkali, alkaline earth, calcium, sodium and ammonium. The solution may also contain natural terpenes in a concentration of 0.5 to 2.0 wt. %, which may also require 0.5 to 2.0 wt. % of a surfactant to solubilize the terpene. Terpenes are thought to help the formaldehyde penetrate the bacterial cell wall during the initial sterilization process.
Feedstuffs referred to in this invention preferably have a moisture content from 5 to 20%. Higher water content provides an ideal environment for subsequent bacterial growth making it difficult to prevent recontamination of the feed. The water content is usually between 6-14 wt. % most preferably below 12%. The moisture level of feed can be determined by measuring the moisture weight loss that occurs during heating of feed at 110xc2x0 C. for 16 hours.
Spray application of the formaldehyde solution to the feedstuffs is done at ambient temperature, which can range widely from winter to summer, but normally is in the range of 5xc2x0 C. to 40xc2x0 C. Subjecting the treated feed to increased temperature in the pelletizing process is acceptable and does not destroy the effectiveness of the treatment.
Vaccines of the present invention include those composed of killed or attenuated (modified live virus) viral particles, which are produced by several companies including Fort Dodge Animal Health, Pfizer, Bio-immune, Merial-Select, ASL, Intervet and Shearing-Plough Animal Health Corp. There are numerous other vaccine products available in the United States and elsewhere. The vaccines which are commercially available and commonly used include vaccines against Marek""s disease, Newcastle disease-infectious bronchitis, laryngotracheitis, avian encephalomyletis, fowl pox, pseudorabies, influenza, transmissible gastroenteritis, porcine reproductive and respiratory syndrome, foot and mouth disease and parvovirus. This list is not all-inclusive.
Bacterins are killed or attenuated bacteria suspended in an aqueous, or oil emulsion or adjuvant type formulation. This type of vaccination is usually administered by subcutaneous, intramuscular or nasal/oral routes. The companies which produce viral vaccines also typically produce bacterins. The bacterins which are commercially available and commonly used include Salmonella enteritidis, Salmonella cholerasuis, Streptococcus suis, Escherichia coil, Mycoplasma galliseptum, Mycoplasma hyopneumonia, Erysipelas, Bordetella, Leptosprias species, Actinobacillus pleuropneumonia and Haemophilis species. This list is not all-inclusive.
Poultry vaccines can be administered in a number of ways. Inactivated or killed-virus vaccines must be given by injection to each individual bird, which is relatively expensive. Mass vaccination with live-virus vaccines is more economical. Poultry can be immunized by administering live-virus vaccines in the drinking water or aerosol. With the drinking-water and aerosol methods, it is possible to vaccinate large numbers of birds without handling each one. The methods, however, are not without pitfalls. Live viruses used in water vaccination produce mild infections and should be given only to healthy birds. Sick or heavily parasitized poultry may not develop protective immunity. If these conditions are present, it is advisable to postpone the vaccination until the flock has recovered. The exact vaccination protocol is given by the manufacturer of each product. In general, chicks are treated as follows.
In general, mature chickens are vaccinated as follows:
First vaccination
ND (B-1) and IB (modified Massachusetts via DW, IO, or IN route only.
4 weeks later (repeat at 3-month intervals)
ND (LaSota), IB (regular Massachusetts or Massachusetts-Connecticut) via DW, IO, or IN route.
In breeder flocks the high antibody level obtained by repeated vaccinations and maintaining the flock on contamination-resistant feed will assure transmission of a uniform parental immunity to the offspring.